The level of production of a protein in a host cell is governed by three major factors: the number of copies of its gene within the cell, the efficiency with which those gene copies are transcribed and the efficiency with which the resultant messenger RNA ("mRNA") is translated. Efficiency of transcription and translation (which together comprise expression) is in turn dependent upon the nucleotide sequences which are normally situated ahead of the desired coding sequence. These nucleotide sequences or expression control sequences define, inter alia, the location at which RNA polymerase interacts (the promoter sequence) to initiate transcription and at which ribosomes bind and interact with the mRNA (the product of transcription) to initiate translation.
Not all such expression control sequences function with equal efficiency. It is thus often of advantage to separate the specific coding sequence for a desired protein from its adjacent nucleotide sequences and to fuse it instead to other expression control sequences so as to favor higher levels of expression. This having been achieved, the newly engineered DNA fragment may be inserted into a higher copy number plasmid or a bacteriophage derivative in order to increase the number of gene copies within the cell and thereby further to improve the yield of expressed protein.
Because over-production of even normally nontoxic gene products may be harmful to host cells and lead to decreased stability of particular host-vector systems, a good expression control sequence, in addition to improving the efficiency of transcription and translation of cloned genes, should also be controllable so as to modulate expression during bacterial growth. For example, the preferred expression control sequences are ones that may be switched off to enable the host cells to propagate without excessive build-up of gene products and then to be switched on to promote the expression of large amounts of the desired protein products.
Several expression control sequences, which satisfy some of the criteria set forth above, have been employed to improve the expression of proteins and polypeptides in bacterial hosts. These include, for example, the operator, promoter and ribosome binding and interaction sequences of the lactose operon of E. coli (e.g., K. Itakura et al., "Expression In Escherichia coli Of A Chemically Synthesized Gene For The Hormone Somatostatin", Science, 198, pp. 1056-63 (1977); D. V. Goeddel et al., "Expression In Escherichia coli Of Chemically Synthesized Genes For Human Insulin", Proc. Natl. Acad. Sci. USA, 76, pp. 106-10 (1979)), the corresponding sequences of the tryptophan synthetase system of E. coli (J. S. Emtage et al., "Influenza Antigenic Determinants Are Expressed From Haemagglutinin Genes Cloned In Escherichia coli", Nature, 283, pp. 171-74 (1980); J. A. Martial et al., "Human Growth Hormone: Complementary DNA Cloning And Expression In Bacteria", Science, 205, pp. 602-06 (1979)) and the major operator and promoter regions of phage .lambda. (H. Bernard et al., "Construction Of Plasmid Cloning Vehicles That Promote Gene Expression From The Bacteriophage Lambda P.sub.L Promoter", Gene, 5, pp. 59-76 (1979)). This invention relates to the last of these expression control sequences.
Bacteriophage A contains three major promoters--P.sub.L, P.sub.R and P'.sub.R. A repressor protein the product of phage gene cI, is known to control the activity of promoters P.sub.L and P.sub.R. The repressor binds to the respective operator regions--O.sub.L and O.sub.R --of these promoters and blocks initiation of transcription from the corresponding promoter. Moreover, due to its autoregulating mode of synthesis (M. Ptashne et al., "Autoregulation And Function Of A Repressor In Bacteriophage .lambda.", Science, 194, pp. 156-61 (1976)), one copy of the cI gene on the chromosome of a lysogenic strain is able to repress fully the P.sub.L or P.sub.R promoters present in a multi-copy plasmid (infra). It should be noted that in systems involving the lac promoter repression of the promoter under non-induced conditions is only partial (K. Itakura et al., supra; D. V. Goeddel et al., supra).
The control exerted by the repressor over promoters P.sub.L and P.sub.R may be altered by modification of the repressor protein or its gene. For example, one mutation is known where the repressor protein is temperature sensitive. When that mutation is employed, the promoters may be activated or inactivated by varying the temperature of the culture and hence the stability of the repressor.
Bacteriophage .lambda. also contains genes N and cro. The N gene is under P.sub.L control. The product of the N gene is known to act as an anti-terminator in bacteriophage .lambda.. Anti-termination is advantageous in overriding transcript termination or slow-down caused by the presence of termination sequences, termination-like sequences or transcription slow-down sequences in the particular DNA sequences that are to be transcribed. Furthermore, polarity effects, introduced by the presence of nonsense codons in the promoter transcript, may be relieved by the N gene product (N. Franklin & C. Yanofsky, "The N Protein Of .lambda.: Evidence Bearing On Transcription Termination, Polarity And The Alteration Of E. coli RNA Polymerase", in RNA Polymerase (Cold Spring Harbor Laboratory) pp. 693-706 (1976)).
The product of the cro gene transcribed from the P.sub.R promoter is known to be a secondary repressor for both promoters P.sub.L and P.sub.R (J. Pero, "Deletion Mapping Of The Site Of The tof Gene Product", in The Bacteriophage .lambda., (Cold Spring Harbor Laboratory), pp. 549-608 (1971); H. Echols, "Role Of The cro Gene In Bacteriophage .lambda. Development", J. Mol. Biol., 80, pp. 203-16 (1973); A. Johnson et al., "Mechanism Of Action Of The cro Protein Of Bacteriophage .lambda.", Proc. Natl. Acad. Sci. USA, 75, pp. 1783-87 (1978)). Because the cro gene product is co-produced along with the desired products of the host-vector combination, the cro gene product's effect on expression from the P.sub.L or P.sub.R promoters tends to increase with time. Therefore, in any system where continued high levels of expression are desired, deletion or inactivation of the cro gene is necessary.
The effectiveness of the P.sub.L promoter for expression of cloned genes has been demonstrated by incorporating the tryptophan (trp) operon of E. coli into phage .lambda.. (N. Franklin, "Altered Reading Of Genetic Signals Fused To The N Operon Of Bacteriophage .lambda.: Genetic Evidence For Modification Of Polymerase By The Protein Product Of The N Gene", J. Mol. Biol., 89, pp. 33-48 (1979); A. Hopkins et al., "Characterization Of .lambda.trp--Transducing Bacteriophages Made In Vitro", J. Mol. Biol., 107, pp. 549-69 (1976)). In this modified phage, the trp genes can be transcribed either from their own promoter or from the P.sub.L promoter. P.sub.L mediated expression was found to be 3-4 times higher than the levels obtained from the homologous trp promoter.
The effect of repressor on P.sub.L mediated expression was also demonstrated in this modified phage. For example, in the absence of repressor, P.sub.L controlled expression of antranilate synthetase (the first enzyme in the trp operon) was 11 times greater than that observed for the enzyme under trp promotion in the absence of trp repressor (J. Davison et al., "Quantitative Aspects Of Gene Expression In A .lambda. trp Fusion Operon", Molec. gen. Genet., 130, pp. 9-20 (1974)). Yet, in the presence of an active cI gene, P.sub.L mediated expression of the enzyme was reduced at least 900-fold. These studies also demonstrated that continued high level of P.sub.L mediated transcription was only possible if the cro gene was not functional in the host.
The problem is that although the above-described .lambda. trp phages demonstrate the utility of the P.sub.L promoter for the expression of inserted genes, the use of such phages is somewhat restricted by difficulties in construction and stable propagation of cro.sup.- acceptor phages. Without such phages, the observed high levels of expression soon drop off as the level of the co-produced cro gene product increases and represses transcription from the P.sub.L promoter.
While the disadvantage of .lambda. phages has been somewhat overcome by cloning the .lambda. control elements on an autonomously replicating plasmid such as Col EI or its derivatives (J. Hedgpeth et al., "Lambda Phage Promoter Used To Enhance Expression Of A Plasmid-Cloned Gene", Molec. gen. Genet., 163, pp. 197-203 (1978)) or by constructing smaller plasmids that incorporate only the .lambda. P.sub.L system (H. Bernard et al., supra), these latter vectors are disadvantaged by the distance between the sites available for insertion of cloned genes and the P.sub.L promoter. For example, in the vectors described by H. Bernard et al., supra., the distance between the sites of gene insertion and the P.sub.L promoter on the vector range from about 300 to about 8600 bases. Moreover, the more commonly used EcoRI and BamHI insertion sites in Bernard et al.'s vectors are not closer than 600 to 1000 bases, respectively, to the P.sub.L promoter. In addition, the effect of the N gene product on transcription of the desired DNA sequences cannot be readily assessed in Bernard et al.'s vectors because the N gene product is encoded on the plasmid itself and is not of chromosomal origin. Finally, in addition to there being no direct evidence that Bernard's vectors afford higher levels of protein expression, there is no teaching in Bernard that his vectors are usefully employed in the expression of eukaryotic gene products in prokaryotic hosts.